Modulation of CRISPR/Cas12a trans-cleavage activity by various DNA-modifying enzymes

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR/Cas) system is receiving increased attention in biological sciences and particularly, trans-cleavage activity of Cas12a, which indiscriminately cuts single-stranded DNA after recognizing target DNA, is widely used for the detection of biomolecules. In the present study, we showed that CRISPR RNA (crRNA) with 11-mer DNA extension at its 3′-end induced higher trans-cleavage activity of Cas12a than crRNA without DNA extension or with 31-mer or longer DNA extension. This finding was then used to modulate the trans-cleavage activity of Cas12a in response to various DNA modifying enzymes that act on the DNA extension of crRNAs. As a result, we demonstrated that the trans-cleavage activity of Cas12a either increased or decreased only in the presence of specific enzymes such as restriction endonuclease, exonuclease, or terminal transferase. These results are the basis for systems that initiate the trans-cleavage activity of Cas12a at desired time points, but also for detection systems in combination with various signaling methods.